Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line.
The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with Kd values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and Bmax values of 97 +/- 4 and 338 +/- 16 fmol/10(6) cells, respectively. Inhibition of [3H](-)-N-methyl-3-quinuclidinyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihidro-6H-pyrido-[2,3-b](1, 4)-benzodiazepine-6-one > hexahydro-sila-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin-stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 microM (0.2-2.8) and 1.1 microM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2-B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms.
Kovacs
I
I
Pharmacology, The University of Arizona, Health Sciences Center, Tucson, Arizona 85724, USA.
Yamamura
H I
HI
Waite
S L
SL
Varga
E V
EV
Roeske
W R
WR
D000595:Amino Acid Sequence; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018721:Muscarinic Agonists / Q000378:metabolism; D018727:Muscarinic Antagonists / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013045:Species Specificity
D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D002217:Carbachol; D000242:Cyclic AMP